Hormone and albumin fusion protein
Biologically active polypeptides comprising a therapeutically active polypeptide fused to human serum albumin or a variant thereof, methods for the preparation thereof, nucleotide sequences encoding such fusion polypeptides, expression cassettes comprising such nucleotide sequences, self-replicating plasmids containing such expression cassettes, and pharmaceutical compositions containing said fusion polypeptides.
1. A recombinant fusion protein comprising hormone and albumin or an albumin variant, wherein (i) said recombinant fusion protein has a higher plasma stability than unfused hormone, (ii) said recombinant fusion protein retains the therapeutic activity of unfused hormone, and (iii) said albumin or albumin variant is located either at the N-terminus or C-terminus of said recombinant fusion protein.
2. The recombinant fusion protein of claim 1 , comprising albumin.
3. The recombinant fusion protein of claim 1 , comprising an albumin variant.
4. The recombinant fusion protein of claim 3 , wherein said albumin variant is a fragment of albumin.
5. The recombinant fusion protein of claim 3 , wherein said albumin variant is a mature form of albumin.
6. The recombinant fusion protein of claim 3 , wherein said albumin variant has a mutation of one or more residues.
7. The recombinant fusion protein of claim 3 , wherein said albumin variant has a deletion of one or more residues.
8. The recombinant fusion protein of claim 3 , wherein said albumin variant has a mutation and deletion of one or more residues.
9. The recombinant fusion protein of claim 3 , wherein said albumin variant has an addition of one or more residues.
10. The recombinant fusion protein of claim 1 , wherein said recombinant fusion protein comprises an N-terminal Methionine.
11. The recombinant fusion protein of claim 1 , wherein said recombinant fusion protein comprises a peptide linker.
12. The recombinant fusion protein of claim 1 , wherein said recombinant fusion protein comprises a secretion signal sequence.
13. The recombinant fusion protein of claim 12 , wherein said secretion signal sequence is the natural leader sequence of hormone.
14. The recombinant fusion protein of claim 1 , wherein said hormone is fused to the N-terminal end of said albumin or albumin variant.
15. The recombinant fusion protein of claim 1 , wherein said hormone is fused to the C-terminal end of said albumin or albumin variant.
16. The recombinant fusion protein of claim 1 , wherein said recombinant fusion protein is expressed by a prokaryotic cell.
17. The recombinant fusion protein of claim 16 , wherein said recombinant fusion protein is expressed by a bacteria.
18. The recombinant fusion protein of claim 1 , wherein said recombinant fusion protein is expressed by a eukaryotic cell.
19. The recombinant fusion protein of claim 18 , wherein said recombinant fusion protein is expressed by an animal cell.
20. The recombinant fusion protein of claim 19 , wherein said animal cell is a CHO cell.
21. The recombinant fusion protein of claim 19 , wherein said animal cell is a COS cell.
22. The recombinant fusion protein of claim 1 , wherein said recombinant fusion protein is expressed by a yeast.
23. The recombinant fusion protein of claim 22 , wherein said yeast is Saccharomyces.
24. The recombinant fusion protein of claim 18 , wherein said recombinant fusion protein is expressed by a fungi.
25. A nucleic acid molecule comprising a polynucleotide encoding the recombinant fusion protein of claim 1 .
26. A nucleic acid molecule of claim 25 , which comprises a heterologous polynucleotide.
27. The nucleic acid molecule of claim 26 , wherein said heterologous polynucleotide is a vector sequence.
28. The nucleic acid molecule of claim 26 , wherein said heterologous polynucleotide is a promoter sequence.
29. The nucleic acid molecule of claim 28 , wherein said promoter sequence is any one selected from the group:
a. a hybrid promoter;
b. a constitutive promoter;
c. a regulatable promoter;
d. a yeast phosphoglycerate kinase (PGK) promoter;
e. a yeast glyceraldehyde-3-phosphate dehydrogenase (GDP) promoter;
f. a yeast lactase (LAC4) promoter;
g. a yeast enolase (ENO) promoter;
h. a yeast alcohol dehydrogenase (ADH) promoter;
i. a yeast acid phosphatase (PHO5) promoter;
j. a lambda bacteriophage P L promoter;
k. a lambda bacteriophage P R promoter;
l. a tryptophan P trp promoter; and
m. a lactose P lac promoter.
30. The nucleic acid molecule of claim 26 , wherein said heterologous polynucleotide is a selectable marker.
31. The nucleic acid molecule of claim 30 , wherein said selectable marker is any one selected from the group:
a. the URA3 gene;
b. geneticin resistance;
c. metal ion resistance; and
d. ampicillin resistance.
32. The nucleic acid molecule of claim 26 , wherein said heterologous polynucleotide is a region for termination of transcription.
33. An isolated host cell comprising the nucleic acid molecule of claim 25 .
34. An isolated host cell comprising the nucleic acid molecule of claim 26 .
35. A method for producing a recombinant fusion protein, comprising:
a. culturing the isolated host cell of claim 33 under conditions suitable to produce the recombinant fusion protein encoded by said polynucleotide; and
b. recovering said recombinant fusion protein.
36. The method of claim 35 , wherein the isolated host cell is a CHO cell.
37. A method for producing a recombinant fusion protein, comprising:
a. culturing the isolated host cell of claim 34 under conditions suitable to produce the recombinant fusion protein encoded by said polynucleotide; and
b. recovering said recombinant fusion protein.
38. The method of claim 37 , wherein the isolated host cell is a CHO cell.
39. A recombinant fusion protein produced by the method of claim 35 .
40. A recombinant fusion protein produced by the method of claim 36 .
41. A recombinant fusion protein produced by the method of claim 37 .
42. A recombinant fusion protein produced by the method of claim 38 .
43. A composition comprising one or more recombinant fusion proteins of claim 1 .